1. Field of the Invention
The present invention includes methods for inducing differentiation, selective enrichment and/or promoting proliferation of regulatory T cells. More particularly, the present invention relates to an adoptive immunotherapy using a composition enriched for a T cell population whose marker is CD4+CD25+ and expresses Interleukin (IL)-10.
2. Description of the State of Art
The immune response is an exceedingly complex and valuable homeostatic mechanism that has the ability to recognize foreign pathogens while avoiding reacting with constituents of our body; that is, we are tolerant of “self”. The initial response to a foreign pathogen is called “innate immunity” and is characterized by the rapid migration of natural killer cells, macrophages, neutrophils, and other leukocytes to the site of the foreign pathogen. These cells can either phagocytose, digest, lyse, or secrete cytokines that lyse the pathogen in a short period of time. The innate immune response is not antigen-specific and is generally regarded as a first line of defense against foreign pathogens until the “adaptive immune response” can be generated. Both T cells and B cells participate in the adaptive immune response. A variety of mechanisms are involved in generating the adaptive immune response. A discussion of all the possible mechanisms of generating the adaptive immune response is beyond the scope of this section; however, some mechanisms which have been well-characterized include B cell recognition of antigen and subsequent activation to secrete antigen-specific antibodies and T cell activation by binding to antigen presenting cells.
B cell recognition involves the binding of antigen, such as bacterial cell wall, bacterial toxin, or a glyco-protein found on a viral membrane to the surface immunoglobulin receptors on B cells. The receptor binding transmits a signal to the interior of the B cell. This is what is commonly referred to in the art as “first signal.” In some cases, only one signal is needed to activate the B cells. These antigens that can activate B cells without having to rely on T cell help are commonly referred to as T-independent antigens (or thymus-independent antigens). In other cases, a “second signal” is required and this is usually provided by T helper cells binding to the B cell. When T cell help is required for the activation of the B cell to a particular antigen, the antigen is then referred to as T-dependent antigen (or thymus-dependent antigen). In addition to binding to the surface receptors on the B cells, the antigen can also be internalized by the B cell and then digested into smaller fragment within the B cell and presented on the surface of B cells in the context of antigenic peptide-MHC class II molecules. These peptide-MHC class II molecules are recognized by T helper cells that bind to the B cell to provide the “second signal” needed for some antigens. Once the B cell has been activated, the B cells begin to secrete antibodies to the antigen that will eventually lead to the inactivation of the antigen. Another way for B cells to be activated is by contact with follicular dendritic cells (FDCs) within germinal centers of lymph nodes and spleen. The follicular dendritic cells trap antigen-antibody (Ag-Ab) complexes that circulate through the lymph node and spleen and the FDCs present these to B cells to activate them.
Another well-characterized mechanism of adaptive immune response to antigens is the activation of T cells by binding to antigen presenting cells such as macrophages and dendritic cells. T cells compose a cell lineage, which plays a central role in the immune system as a system of biophylaxis against various pathogens. T cells are largely classified into CD4+ T cells and CD8+ T cells. In particular, the former T cells can be classified, depending on cytokine-producing patterns at certain differentiation and maturation stages after stimulation with antigens, into, for instance, Th1 cells producing mainly IFN-γ and Th2 cells producing IL-4. Generally, the former and the latter T cells are deeply involved in biophylaxis as cellular immunity and as humoral immunity, respectively. The immune response is responsible for eliminating pathogens and acquiring resistance to infection based on a delicate balance resulting from functions of such T cells with varied characteristics. It is known that in the normal immune response, a mechanism works to eliminate foreign nonself antigens, but the mechanism does not eliminate autoantigens, which make an organism because of the established immunologic tolerance. However, overresponse of the immune system against autoantigens causes a so-called autoimmune disease. As described above, immunological tolerance against autoantigens is not an absolute mechanism. The mechanisms by which various immunological tolerances are induced are known in a T cell level. One of such mechanisms, called central tolerance, eliminates autoreactive T cell clones in the thymus (Kisielow, P., et al., Nature, 333:742-746 (1988)); and another mechanism, called peripheral tolerance, controls autoreactive T cells outside the thymus. Known to be included in the latter mechanism are induction of cell death or of anergy against self-antigens (Rocha, B., et al., Science, 251:1225-1228 (1991); Jenkins, M. K., et al., J. Exp. Med., 165:302-319 (1987)), and active suppression by regulatory T cells (Shevach, E. M., Annu. Rev. Immunol., 18:423-449 (2000)). The regulatory T cell is a new, recently proposed concept of T cells, and is defined as having a suppressive action against other T cells. The immune response is operated based on a delicate balance. For example, the above Th1 cells and Th2 cells function antagonistically to their respective immune response, and it has become known that one acts as regulatory T cells on the other. However, verification of the presence of a cell population as regulatory T cells and property analysis thereof has been a point of considerable debate throughout the recent history of immunology. Such regulatory T cells have been studied in vitro or in vivo as cells capable of suppressing or regulating certain immune responses, so that the T cells have been reported as different cell populations according to the cell surface markers, the types of cytokines produced, suppressive and regulatory mechanisms and the like (Roncarolo, M. G., et al., Curr. Opinion. Immunol., 12:676-683 (2000)).
The most studied cell population among these regulatory T cells is a T cell population whose marker is CD4+CD25+ described below. This T cell population has been mainly studied in species of non-human organisms, such as mice and rats. The property analysis of the T cell population has been performed using as an index the fact that organ-specific autoimmune diseases (for example, thyroiditis, insulin-dependent diabetes mellitus, colitis) are induced by transferring T cells, from which certain T cells have been removed using expression of a particular cell surface molecule as an index, into T cell- and B cell-deficient SCID mice or rats (Sakaguchi, S., et al., J. Exp. Med., 161:72 (1985); Itoh, M., et al., J. Immunol., 162:5317-5326 (1999)). Specifically, CD25+, RT6.1+ (expressed in most mature T cells in rat), CD5 highly positive, or CD45RB weakly positive (mice) or CD45RC weakly positive (rats) cells are removed from the CD4+ spleen cells of normal mice or rats, and then the remaining T cells are transferred to T cell- and B cell-deficient SCID mice or rats, thereby inducing organ-specific autoimmune diseases. To date, no such regulatory T cell-specific marker has been observed. That is, the above marker cannot be directly related to the function of regulatory T cells and it represents merely a state of cells being activated, that of cells being stimulated with antigens, or that of cells being immunological memory. However, the regulatory T cell population has been further analyzed using as an index the fact that the cell population is capable of suppressing autoimmune disease and autoimmune inflammation when a certain cell population is transferred, in addition to being capable of inducing organ-specific autoimmune disease in immunodeficient animals (Itoh, M., et al., J. Immunol., 162:5317-5326 (1999); Sakaguchi, S., et al., J. Immunol., 155:1151-1164 (1995); Asano, M., et al., J. Exp. Med., 184:387-396 (1996); Read, S., et al., J. Exp. Med., 192:295-302 (2000); Salomon, B., et al., Immunity, 12:431-440 (2000); Stephens, L. A., et al., J. Immunol, 165:3105-3110 (2000)). Therefore, it is now known that a CD4+CD25+T cell population is capable of use as a marker of the regulatory T cells, conventionally.
Though CD4+CD25+ regulatory T cells have been identified in mice and rats as described above, several groups just recently reported in 2001 the presence of similar cells in humans (Jonuleit, H., et al., J. Exp. Med., 193:1285-1294 (2001); Levings, M. K., et al., J. Exp. Med., 193:1295-1301 (2001); Dieckmann, D., et al., J. Exp. Med., 193:1303-1310 (2001); Taama, L. S., et al., Eur. J. Immunol., 31:1122-1131 (2001); Stephens, L. A., et al., Eur. J. Immunol., 31:1247-1245 (2001); Baecher-Allan, C., et al., J. Immunol., 167:1245-1253 (2001)). The basis of these reports is that a cell population isolated from human peripheral blood, when expression of CD4 and CD25 known for mice is used as an index, has properties equivalent to those reported for mice, in terms of various cell surface markers, anergy of cells to stimulation for activation, types of cytokines produced, in vitro proliferation inhibitory function of normal T cells, the mechanism thereof, and the like. Specifically, CD4+CD25+ T cells isolated from human peripheral blood express CD45R0+ memory T cell markers, and compared to CD4+CD25− T cells, highly express activation markers such as HLA-DR. Further, CD4+CD25+ T cells constantly express CTLA-4 within the cells, and the expression of CTLA-4 is enhanced by stimulation. Furthermore, some stimulations such as stimulation with anti-CD3 antibodies, stimulation with anti-CD3 antibodies and anti-CD28 antibodies, stimulation with allogeneic mature dendritic cells (allogeneic mature DC) do not cause CD4+CD25+ regulatory T cells to synthesize DNA and to produce cytokines. That is, CD4+CD25+ regulatory T cells are in an anergic state (anergy) following stimulation with antigens. Stimulation with cytokines, such as IL-2, IL-4, IL-15, in addition to that with anti-CD3 and anti-CD28 antibodies enhance the ability of CD4+CD25+ regulatory T cells to synthesize DNA, but the ability is not comparable to that of CD4+CD25− T cells. When CD4+CD25− T cells are stimulated with anti-CD3 antibodies or allogeneic mature DC in the presence of CD4+CD25+ regulatory T cells, in comparison with that in the absence of CD4+CD25+ regulatory T cells, proliferation inhibitory action is observed in a CD4+CD25+ regulatory T cell number-dependent manner. CD4+CD25+ regulatory T cells have ability to produce suppressor cytokines, such as IL-10 and TGF-β-1, which is lower than that of mice. However, it has been reported that the proliferation inhibitory action against CD4+CD25− T cells is not canceled by neutralizing antibodies against these cytokines and the inhibitory action requires direct intercellular contact between CD4+CD25− T cells and CD4+CD25+ regulatory T cells. Though the presence of CD4+CD25+ regulatory T cells in mice, rats and humans has been reported, and the property is being analyzed, detailed mechanisms of differentiation and suppressive action of these cells are still in the process of being elucidated, and no specific marker has been found so far.
Moreover, regulatory T cells, which are induced in a mouse and a human by repeated stimulation with allogeneic antigens or allogeneic immature DC in the presence of IL-10 have been also reported (Groux, H., et al., Nature, 389:737-742 (1997); Jonuliet, H., et al., J. Exp. Med., 192:1213-1222 (2000)). Unlike Th1 and Th2 cells, these cells called Tr1 cells are characterized by producing high levels of IL-10, moderate levels of TGF-β-1, IFN-γ and IL-5, low levels of IL-2, and no IL-4. Similar to CD4+CD25+ regulatory T cells, Tr1 cells are anergic, and the T cell-suppressive mechanism can be partially explained by the IL-10 and TGF-β1 produced. However, whether Tr1 cells and CD4+CD25+ regulatory T cells are T cell subsets, which are totally different from each other, or are the same cells but which differ in their differentiation activation stage remains unknown.
Using expression of regulatory T cell markers CD4 and CD25, known among mice and rats, as an index, CD4+CD25+ T cells have been isolated from human peripheral blood. Thus, the isolated T cells have been confirmed to share similar functions with other known cell surface markers of mice or rats, suggesting the presence of CD4+CD25+ regulatory T cells in humans.
These T cells are of a rare cell population, which accounts for merely 5 to 10% of CD4+ T cells of peripheral blood, and are anergic to stimulation for activation and proliferation. In this case, cell proliferation can be promoted by stimulating with cytokines, such as IL-2, IL-4 and IL-15, in addition to anti-CD3 antibodies and anti-CD28 antibodies. However, this is not at a sufficient level for clinical applications, such as an application that involves increasing cell count and transferring the cells into a human.
Since regulatory T cells act suppressively on autoimmune disease, transplant rejection, graft versus host disease (GVHD) when transferred into an animal (Hara, M., et al., J. Immunol., 166:3789-3796 (2001); Taylor, P. A., et al., J. Exp. Med., 193:1311-1317(2001)), it is considered that regulatory T cells may be applied to cellular medicine using their immunosuppressive action to treat autoimmune disease, transplantation or the like. Development of a pharmaceutical composition which promotes proliferation of regulatory T cells, or development of a therapy which involves treating ex-vivo peripheral blood or myeloma cells collected from patients or volunteers, allowing regulatory T cells to proliferate, and returning the cells into the bodies of patients, is being considered.
Pathogens
A recent report suggested that Helicobacter hepaticus infection may result in the induction of regulatory T cells that prevent bacteria-induced colitis (Kullberg, M. C., et al., The Journal of Experimental Medicine, 196:505 (2002)). It was hypothesized that the induction of these cells in response to gut flora may be a protective mechanism to limit tissue damage associated with inflammatory bowel disease. Conversely, during infection by Leishmania major, regulatory T cells accumulate in the skin, where they suppress the ability of effector T cells to eliminate the parasite from the site (Belkaid, Y., et al., Nature, 420:502 (2002)). The latter data suggested that the induction of regulatory T cells by a pathogen may constitute a virulence mechanism by which this organism suppresses and evades the host response.
Actinobacillus actinomycetemcomitans is a pathogenic bacterium with potent cytolytic potential. This pathogen has been implicated in a number of diseases, including periodontitis (Fives-Taylor, P. M., et al., Periodontology, 20:136 (2000)), as well as in a number of non-oral infections, e.g., cardiovascular, intracranial, thoracic and skin infections (van Winkelhoff, A. J., et al., Periodontology, 20:122 (2000)). Infections with A. actinomycetemcomitans are difficult to eradicate (Mombelli, A., R., et al., Journal of Periodontology, 65:827 (1994); Mombelli, A., et al., Journal of Periodontology, 65:820 (1994); Mombelli, A., et al., Journal of Periodontology, 71:14 (2000)). In chronic and aggressive forms of periodontitis, A. actinomycetemcomitans can persist as a chronic infection that may span years to decades (Slots, J., et al., Journal Of Dental Research, 63:412 (1984)). The underlying mechanisms responsible for the inability of the host to eliminate this infection and for the persistence of this organism have not been determined.
As discussed previously, failure of immunologic self-tolerance often leads to the development of autoimmune disease, which is estimated to afflict up to 5% of the population. Although the etiology of autoimmune disease is at present largely unknown, it is will documented that T cells are the key mediators of many autoimmune diseases, such as but not limited to inflammatory myopathy, Myasthenia Gravis, inflammatory polyneuropathies, Multiple Sclerosis, asthma, insulin-dependent diabetes mellitus (IDDM), autoimmune thyroiditis, autoimmune gastiritis accompanying pernicious anemia, and colitis.
There remains an urgent need to provide means to suppress the immune system using safe compositions that can be repeatedly administered, and which are effective to prevent and/or treat diseases amenable to treatment by supression of an immune response such as autoimmune diseases, transplant rejections, and cancer.